Localization of eukaryotic initiation factor 3 on native small ribosomal subunits

Abstract

Te localization of eukaryotic initiation factor 3 (eIF-3) on native small ribosomal subunits has been established by electron microscopy through a comparison of native small ribosomal subunits with derived subunits and with native subunits stripped of eIF-3. Small subunits derived from reticulocyte ribosomes by the puromycin/KCI method are seen in electron micrographs as elongated particles, divided by a heavily stained partition into approximately one-third and twthird domains. Most particles (60-70%) observed in electron micrographs of native small subunit preparations resemble derived small subunits, but have an additional mass attached to one side, thus producing profiles with a three-lobed appearance. The mass measures approximately 160 X 100 X 60 A, and its particle weight is estimated to be about one-third to one-half tat of a 40S subunit. The site of attachment of the additional mass is located on a prominence extending from the central part of the small subunit and is separated by a cleft from the smaller third of the subunit. The remaining particles in preparations of native subunits resemble the profiles seen in electron micrographs of derived subunits. After removal of eIF-3 by treatment with high concentrations of salt, profiles observed in electron micrographs of washed, native subunits were indistinguishable from those of derived subunits. Since removal of eIF-3 coincided with removal of a mass of the correct molecular weight, subunits with the three-lobed appearance are identified as native small subunits carrying eIF-3.Preparations of native small ribosomal subunits from rabbit reticulocytes are able to carry out the initiation steps of protein synthesis when added to an in vitro system containing globin mRNA, derived large ribosomal subunits, and the components of the pH 5 enzyme fraction (1, 2). This ability is due to the presence of initiation factors, particularly to a large protein complex consisting of at least 10 polypeptides (3) which is attached to the native subunits. When isolated, the large protein complex corresponds in its sedimentation value and in the molecular weights of its components to eukarytoic initiation factor (eIF-3) (3-8). The large S value (15-17 S) and the estimated molecular weight (greater than 500,000) of the complex prompted us to attempt its localization by electron microscopy in preparations of native small ribosomal subunits. METHODSPreparation of Native Small Ribosomal Subunits. Native small ribosomal subunits were prepared from rabbit reticulocytes as described (1). In brief, blood from anemic rabbits (9) min at 20,000 X gay was recentrifuged twice: first for 45 min to remove polysomes and ribosome monomers and then for 5 hr at 105,000 X gay in a Ti6O rotor. The 5-hr sediment, containing the bulk of the native ribosomal subunits, was resuspended in solution A at a concentration of 120 A20 units/ml; 0.5-ml aliquots were layered onto 12 ml of 10%-40% sucrose density gradients containing 20 mM triethanolamine-HCI, pH 7.5/90 mM KCI/3 mM MgCl2/2 mM dithi...

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association

“…eIF2-ternary complex also binds to the interface side of the 40S subunit (Bommer et al 1991) and could therefore enhance eIF3's activity in preventing subunit reassociation in a similar manner. Although it has been suggested that eIF3 is attached to the back lobes of the 40S subunit, oriented away from the intersubunit interface, which would necessarily imply that eIF3's dissociation/antiassociation activity is due to induced conformational change rather than to steric hindrance (Srivastava et al 1992;Válasek et al 2003), this contradicts other studies (Emanuilov et al 1978;Lütsch et al 1986). The mechanism by which eIF3 dissociates ribosomes therefore remains an open question.…”

“…Although biophysical data on the location of eIF3 bound to the 40S subunit are contradictory (Emanuilov et al 1978;Lütsch et al 1986;Srivastava et al 1992), biochemical data (Válasek et al 2003) support a model in which eIF3 binds to the solvent side of the 40S subunit, where the eIF3's and 40S subunit's mRNA-binding surfaces could either dock together to form a mRNA-binding tunnel or eIF3 by itself could extend the mRNA-binding channel of the 40S subunit outside the interface surface.…”

Specific interaction of one subunit of eukaryotic initiation factor eIF-3 with 18S ribosomal RNA within the binary complex, eIF-3.small ribosomal subunit, as shown by cross-linking experiments

“…Electron microscopic analysis of native 40S subunits suggests that eIF-3 is located in the neck region of the subunit close to the ribosomal subunit interface [3]. In the present experiments we have shown by chemical cross-linking with the bifunctional reagent DEB and the heterobifunctional reagent ABAI that the 66 000 dalton subunit of eIF-3 is covalently attached to 18S rRNA in the binary 40S-eIF-3 complex.…”

Structure of 18 S Ribosomal RNA in Native 40 S Ribosomal Subunits

“…Emanuilov et al (28) found that the binding site of eIF-3 on 40 S N subunits was partly overlapping the interface region of the particle, suggesting that the ribosome-bound factor directly interferes with subunit joining. However, others have found that eIF-3 binds to the 40 S subunit without interfering with the interface (29,30) (Fig.…”